My long term goal is to understand the effects of macromolecular crowding on biochemical processes by acquiring atomic-level information on proteins under actual biological conditions. To this end, my group has pioneered in-cell NMR. We have shown how to assess structure, quantify dynamics, and measure stability under the crowded conditions found in living Escherichia coli cells using this powerful new technique. Now, we want to take the next step. With support from a Pioneer Award, we will focus on using in-cell NMR in eukaryotic cells to study two key proteins in neurodegenerative diseases, the intrinsically disordered proteins, a-synuclein and tau. These proteins are excellent candidates not only because of their disease relevance, but also because we know that macromolecular crowding has extremely large effects on the properties of disordered proteins. Our understanding of protein structure and function has grown enormously in the last 100 years. We have progressed from pondering what role, if any, polypeptides play in the cell, to unraveling, at the atomic level, the mechanisms of enzymes and the molecular bases of protein-protein interactions vital to understanding human disease. Our accumulating wealth of knowledge has largely come from in vitro studies performed under conditions far different from those found in biology. For example, most biochemical examinations of protein behavior are performed at concentrations in the ?g-to-mg-per-mL range, but the insides of cells, where most proteins perform their work, have protein concentrations of >300 mg per mL. Thus, our knowledge comes from data acquired under conditions that are far from physiological relevant, and theory predicts these differences can have extremely large effects on biophysical parameters. Moving beyond the test tube by performing truly in vivo studies in living eukaryotic cells by using NMR spectroscopy is the next frontier in protein chemistry.